The DNA untwisting enzyme introduces a transient nick into double-stranded DNA and thereby provides a swivel for the strands of the helix to wind relative to one another. Winding of the helix will occur providing that some force exists within the molecule (e.g. superhelical turns in a circular DNA) to drive the winding process. One of the objectives of this research proposal is to study the mechanism of the reaction catalyzed by the untwisting enzyme. Since the nicking-sealing reaction sequence occurs in the absence of energy-donating cofactors, the energy of the phosphate ester linkage must be conserved during the lifetime of the nicked intermediate. Using enzyme purified from rat liver nuclei, an attempt will be made to identify and isolate the nicked intermediate. An examination of the structure of the two termini at the nick, particularly with respect to the possible covalent attachment of an energy rich moiety, may reveal the mechanism of the reaction. A second objective is to inquire about the functional role of the DNA untwisting enzyme in the cell. We have previously suggested that the enzyme may provide the swivel for the DNA strands to unwind during the replication process. The direct approach to this problem will be to screen temperature sensitive yeast mutants for an altered or heat-labile untwisting enzyme. The phenotype of such mutants at the nonpermissive temperature may suggest a role for the enzyme in the cell. The second approach will be to study the regulation of the untwisting enzyme during the cell cycle and as a function of the growth rate of cells. A positive correlation of any regulatory patterns observed for the untwisting enzyme with the regulation of other enzymes suspected to be involved in DNA synthesis, would suggest an involvement of the enzyme in the replication process. Alternatively, constant enzyme levels during the entire cell cycle or independent of the growth state of the cells, would suggest a multifunctional role for the enzyme.